The main objective of the research proposed here is to discover and quantitate the segmental motions of cardiac and skeletal sacroplasmic reticulum Ca2-ion/Mg2-ion-ATPase during Ca2-ion transport. Although conformational changes have been demonstrated for this ATPase, none of the techniques used can distinguish local changes from global ones (i.e., segmental motions). Such motions would be measured by fluorescence energy transfer (FET) between donor and acceptor fluorophores attached to unique sites on different segments of the enzyme. FET is sensitive to changes in distance between 0 and 5 nm and therefore suitable for the movements involved in transporting Ca2-ion across an 8 to 10 nm thick membrane. The SR ATPase can be trapped in intermediate states along the Ca2-ion transport reaction path by manipulating the conditions. In this way the mechanical motions of the calcium pump can be correlated to the energetics of the ATP binding and hydrolysis steps which yield the phosphorylated enzyme intermediate. The relative contributions of each to energy transduction is one of general interest. The method of assuring proper labeling will be limited proteolysis followed by radiographic gel electrophoretical analysis and peptide mapping.